An ampouled preparation of bacterial endotoxin, coded 84/650, was evaluated in 35 laboratories in 12 countries for its suitability to serve as the International Standard for Endotoxin. The ampouled preparation was calibrated in terms of the USA National Standard, EC5, in Limulus Amoebocyte Lysate gelation, turbidimetric and chromogenic tests and in rabbit pyrogen tests. On the basis of the results reported here, with the agreement of the participants in the study, and with the authorization of the Expert Committee on Biological Standardization of the World Health Organization, the preparation coded 84/650 was established in 1986 as the International Standard for Endotoxin for Limulus Gelation Tests with an assigned unitage of 14,000 IU of endotoxin per ampoule.
Recent advances in methods for the manufacture of inactivated poliovirus vaccines have resulted in increased vaccine immunogenicity. In conjunction with this capability it is important to have available highly sensitive and quantitative potency assays. The potential suitability of enzyme-linked immunoassay (ELISA) was evaluated using animal sera with neutralizing antibodies or neutralizing monoclonal antibodies for antigen detection in potency tests. The monoclonal antibodies developed, which bound D antigen but not C antigen, were neutralizing unless relatively weakly reactive. Those that bound C antigen only were non-neutralizing. Those that bound both C and D antigens were sometimes neutralizing. D-specific and D/C-specific neutralizing monoclonal antibodies against type-2 poliovirus protected mice on passive immunization against paralytic disease and death from the MEF strain virus. Potency measurements by ELISA using either D-specific neutralizing monoclonal antibodies or type-specific goat sera for antigen detection were sensitive and precise. Tests using C-specific monoclonal antibodies for antigen detection indicated that increased C antigen content may result in falsely elevated reactivities of animal sera with some vaccines. Monoclonal antibodies may be useful ELISA reagents for IPV potency testing.
The in vitro response of human B- and T-lymphocytes to the acellular vaccines JNIH-6 (containing pertussis toxoid and filamentous hemagglutinin), and JNIH-7 (containing pertussis toxoid), and to the purified components JNIH-4 (filamentous hemagglutinin) and JNIH-5 (pertussis toxin) was investigated. Pertussis toxoid and filamentous hemagglutinin induced specific Ig synthesis in vitro in lymphocytes obtained from convalescent pertussis patients as target cells. The antigen-dependent Ig production was demonstrated in lymphocyte culture supernatants by ELISA techniques and by a chinese hamster ovary cell toxin neutralization assay. Particularly with JNIH-4, -6 and -7, high antibody titers were obtained. At optimal antigen concentrations a marked lymphocyte blast transformation was found in lymphocyte cultures from whooping cough patients, but not in cultures of lymphocytes obtained from healthy volunteers. At high concentrations native pertussis toxin as well as the B oligomer (S2-5) of the toxin induced a strong proliferation of patient as well as control lymphocytes, indicating non-specific mitogenic activity. At lower concentrations lymphocyte blast transformation was seen in patient cultures only, which indicates an antigen-specific T-cell response. The A protomer (S1), dimer 1 (S2 + 4) and dimer 2 (S3 + 4) induced proliferation of patient lymphocytes, which demonstrates the presence of T-cell epitopes on these peptides. The in vitro B-cell response and the lymphocyte blast transformation assay are both useful tools for estimating the potency of acellular pertussis vaccines in man. Spontaneously acquired and vaccine induced immunity to Bordetella pertussis can be investigated at the level of B- and T-lymphocytes.
A new tick-borne encephalitis (TBE) vaccine for human use has been developed. TBE virus (TBEV) was propagated in primary chick embryo cells, inactivated by formalin and purified by continuous-flow density gradient centrifugation. The TBE vaccine was tested for innocuity, immunogenicity and protective capacity in a series of laboratory tests. The results indicated that the vaccine is outstandingly well tolerated, highly immunogenic in various laboratory animals, and induces protective immunity in mice. These data suggest that this new vaccine should be studied in clinical trials.
A reference preparation for opacity consisting of a plastic rod was introduced by Perkins et al. in 1973. It was adopted as the International Reference Preparation for Opacity in 1975. This plastic rod opacity reference preparation has been used to standardize the Chinese National Bacterial Opacity Standard. The material was prepared from plastic sheet by a water-bath method and by a dry-heat method; the sheet was then machined into the plastic rods. We have studied the technical processes and set up methods for the examination of the sheets and rods. The water-bath method was found to be better than the dry-heat method in our tests. Collaborative assays in research institutes of biological products have shown that the plastic rod can replace the glass-powder suspension. The duration of validity of the plastic rod opacity reference preparation and that of the glass-powder suspension used for the Chinese National Bacterial Opacity Standard were studied and found to be similar. For this reason the plastic rod opacity reference preparation has not been widely used in China.
Dans les concentrats proteiques provenant de plasma humain, HIV-1 est detruit par la chaleur apres pasteurisation. HIV-2 est detruit apres 10 minutes de chauffage a 60 o C, ainsi que HIV-1, le virus RSV etant nettement moins thermosensible. Quoi qu'il en soit, les retrovirus, par cette methode, sont inactives apres un temps de chauffage beaucoup plus court que les 10 heures employees dans le procede industriel
Leningrad-L3 Mumps Vaccine virus has been further attenuated by adaptation and passage on SPF chick embryo fibroblast cell cultures. This new mumps strain has been designated L-Zagreb and has been used to prepare mumps vaccines which meet the WHO requirements. Observations during both the field trial period prior to registration and during the later use of the vaccine showed that the few reactions observed were mild and that seroconversion was obtained in 88-98% of vaccines. The morbidity of mumps in Croatia declined more than tenfold after the introduction of the new vaccine. During a mumps epidemic, vaccine efficiency was calculated to be 97-100%.
Soixante sept anatoxines tétaniques ou dipthériques, deux toxines: une tétanique et une diphtérique, deux vaccins: un DTP et un DPTP ont été étudiés par électrofocalisation en gel d’agarose. Beaucoup de fractions (6 à 15) ont été mises en évidence dans ces produits qui montrent une grande diversité d’un producteur à l’autre et même, dans certains cas, d’un lot à l’autre d’un même producteur, en particulier dans le cas des anatoxines diphtériques. Ceci pourrait être utilisé dans la recherche sur ces vaccins ainisi que pour le controle (de production ou national), particulièrement pour vérifier la régularité des lots successifs.
A study has been carried out in the Ivory Coast to assess the efficacy of a combined vaccine against yellow fever and measles relative to that of each vaccine administered separately. Healthy children aged six to nine months were recruited and divided into two age groups: less than seven months (group I) and more than eight months (group II). In each group, they were randomly assigned to receive either yellow fever vaccine only (A), measles vaccine only (B), or the combined vaccine (C). The serological responses to measles and yellow fever were assessed in 219 initially seronegative children 45 days after immunization. More than 90% of the children developed yellow fever haemagglutination inhibiting antibodies. Neither age nor combination with measles vaccine influenced the responses to yellow fever vaccine. Measles haemagglutinational inhibiting antibodies were found in 97% of the children and the seroconversion rate was influenced neither by age nor by combination with yellow fever vaccine. Younger infants had lower titres of measles antibody. No particular adverse reactions were notified during the follow up. This study shows that combined yellow fever and measles vaccines are immunogenic in infants from the age of six months. Controlling yellow fever in endemic areas and the prevention of measles in young infants may greatly benefit by this combination.
Lipopolysaccharide (LPS, i.e. endotoxin) present in meningococcal outer-membrane protein and polysaccharide preparations made for vaccine use was quantitated by a silver-stain method following SDS-PAGE. The reactivities of LPS in the preparations were also measured by rabbit pyrogenicity and Limulus amoebocyte lysate (LAL) assay. Although rabbit pyrogenicity and LAL assay are more sensitive than the silver stain method, the latter provided an actual amount of LPS present in the protein or in the polysaccharide. For a meningococcal protein preparation, rabbit pyrogenicity showed about one-tenth, and even less by LAL assay, of the actual amount of LPS. This is because protein-bound LPS in meningococcal protein preparations is about 10-fold less active in causing fever in rabbits, and 20- to 40-fold less active in the gelation of LAL than the same amount of a purified free LPS which is generally used as a reference in quantitating LPS in these two assays. As for the small amount of LPS present in a meningococcal polysaccharide preparation, similar LPS content was obtained when measured by the three methods suggesting that the LPS is not bound to the polysaccharide in contrast to that in the proteins mentioned above. The purified meningococcal LPS was pyrogenic in rabbits at 1 ng/kg.
The use of the principle of inhibition of toxin binding to an antitoxin coated immunoassay plate as described in a previous paper for tetanus antitoxin titration, was adapted for the estimation of diphtheria antitoxin in human sera. With a few modifications, a Toxin-Binding Inhibition (ToBI) test was developed which could be used for a combined estimation of both tetanus and diphtheria antitoxin levels. The application of streptavidin-biotinylated peroxidase complex when using small serum samples (less than 50 microliters) is discussed. Antitoxin titres (both diphtheria and tetanus) of 0.002 IU ml-1 were detectable by the ToBI test, this being far below the level considered to be protective in man. Sera from 140 adults with different vaccination histories were titrated for both tetanus and diphtheria antitoxin. Good correlations were found between the estimates obtained by the ToBI test and those obtained by the toxin-neutralization (TN) test in mice (tetanus antitoxin) and those obtained in the in vitro neutralization test in VERO cells (diphtheria antitoxin). It is concluded that the ToBI test is a simple and reliable alternative to the functional models currently in use for the estimation of diphtheria and tetanus antitoxin levels. In addition, the ToBI test eliminates the need for laboratory-animal or cell-culture facilities and can be performed with small quantities of serum as required in field trials.
This paper reports a comprehensive study of the biological stability of an immunoaffinity purified preparation of human thyroid stimulating hormone (TSH) and provides a reference against which future natural or synthetic preparations may be compared. The stability of the hormone preparation was investigated using the accelerated degradation method. The bioassay of the TSH was carried out in mice using a modified McKenzie method. Analysis of the results showed that the preparation was as stable as other TSH preparations purified by conventional methods.
A collaborative study was conducted to establish a suitable international reference reagent for hepatitis B vaccine for use in immunogenicity assays. The limiting dilution required to induce antibodies in 50% of the test animals was determined for the proposed international reference reagent and three other plasma-derived hepatitis B vaccines. The minimum antigenic dose of these preparations varied widely (100-fold range) between laboratories. However, the expression of potencies of vaccines relative to the proposed International Reference Reagent reduced the variation between laboratories to within a 10-fold range. The reference reagent is intended for use in assays of hepatitis B vaccines in mouse (or guinea-pig) immunogenicity studies. For products made by different procedures, clinical trials in humans are necessary to establish a correlation between the immunogenic potency in animals and man.
A simple, indirect enzyme immunoassay using purified enterovirus adsorbed on to nitrocellulose has been developed for screening monoclonal antibodies to enteroviruses. The sensitivity of the assay ranged from 10 ng to 1 microgram of viral protein and was 10- to 50-fold more sensitive than conventional EIA on microplates. This simple, sensitive and specific assay proved to be a useful and practical tool for detecting monoclonal antibodies which would not be found in a conventional EIA screening procedure.
In comparison with the presently used potency test for diphtheria vaccine, in vitro examination of the immunogenicity of the vaccine would have great advantages. For this reason in vitro induction of diphtheria toxoid specific antibody synthesis in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid was investigated. The results showed that a dose dependent synthesis of diphtheria antibody was induced by adsorbed diphtheria toxoid and combined vaccines containing the diphtheria toxoid component. Plain diphtheria toxoid appeared to be less immunogenic in comparison with adsorbed toxoid. There is some indication that the pertussis component had a stimulating effect on the diphtheria antibody synthesis. In conclusion, these results are promising for in vitro examination of the immunogenicity of diphtheria vaccines. The model will be validated for the routine control of diphtheria vaccine.
